Structure-Function Analysis of the Gamma Carboxylase

描述（由申请方提供）：维生素K依赖性（VKD）羧化酶对止血至关重要，因为它将VKD蛋白中的Glus转化为羧化Glus，使其与发生止血的细胞表面结合。VKD蛋白在其分泌期间在内质网中被羧化，并且单个羧化酶修饰所有VKD蛋白，其中许多在组织中共表达。在上一个资助期，我们开发了一种方法来直接分析哺乳动物细胞中的细胞内羧化，这表明分泌过程影响羧化，细胞内处理是不相同的所有VKD蛋白和羧化是由谷氨酸羧化所需的减少维生素K辅因子的可用性。研究还表明，VKD周转的限速步骤在细胞中与体外反应中不同，这可能是由于羧化酶的翻译后修饰。其他研究表明，导致钩端螺旋体病的细菌病原体钩端螺旋体含有VKD羧化酶的直系同源物，该酶似乎是通过水平基因转移获得的，并且已经适应了羧化以外的作用。我们的研究还涉及新的功能性羧化酶残基，包括启动羧化的催化碱基和取代导致组合VKD凝血因子缺乏的残基。我们的长期目标是了解羧化的机制，包括它如何与分泌机制相互作用，以及多种VKD蛋白如何被一种羧化酶修饰，使其完全羧化并具有活性。我们建议：1.确定活性部位如何促进羧化。我们将确定催化基地，启动羧化，并将确定如何在羧化酶残基的取代导致合并VKD因子缺乏症。2.确定VKD蛋白的周转是否不同，是否受到VKD蛋白-羧化酶相互作用的第二个位点的影响。我们将确定VKD蛋白因子X和凝血酶原是否以相同的效率被羧化，以及具有VKD蛋白-羧化酶相互作用的两个位点是否影响效率。3.为了验证我们的假设，即翻译后羧化酶修饰对VKD蛋白质周转很重要。将鉴定羧化酶中的翻译后修饰位点并使其突变，以确定其是否影响VKD蛋白质周转。这些研究将为理解羧化做出重要贡献，这对于开发上级抗凝剂和生产用于血友病和脓毒症治疗的VKD蛋白具有重要意义。抽象点。饮食中的维生素K用于激活一系列对血液凝固至关重要的因素，因此了解它们是如何被激活的非常重要。这些研究将影响抗凝剂的开发和治疗血友病和感染性休克的治疗性蛋白质的生产。

DESCRIPTION (provided by applicant): The vitamin K-dependent (VKD) carboxylase is critical to hemostasis because it converts Glus to carboxylated Glus in VKD proteins to allow their binding to cell surfaces where hemostasis occurs. VKD proteins are carboxylated in the endoplasmic reticulum during their secretion, and a single carboxylase modifies all VKD proteins, many of which are coexpressed in tissue. In the previous grant period, we developed an approach to directly analyze intracellular carboxylation in mammalian cells, which showed that the secretory process impacts carboxylation, that intracellular processing is not identical for all VKD proteins and that carboxylation is regulated by the availability of the reduced vitamin K cofactor required for Glu carboxylation. The studies also showed that the rate-limiting step in VKD turnover is different in cells than in an in vitro reaction, which may be due to post-translational modifications in the carboxylase. Other studies revealed that Leptospira, the bacterial pathogen that causes leptospirosis, contains an ortholog of the VKD carboxylase, which appears to have been acquired by horizontal gene transfer and to have been adapted for a role other than carboxylation. Our studies also implicated novel functional carboxylase residues, including the catalytic base that initiates carboxylation and residues whose substitution cause combined VKD coagulation factor deficiency. Our long-term goal is to understand the mechanism of carboxylation, including how it interfaces with the secretory machinery and how multiple VKD proteins are modified by one carboxylase to become fully-carboxylated and active. We propose: 1. To determine how the active site facilitates carboxylation. We will identify the catalytic base that initiates carboxylation and will determine how substitutions in carboxylase residues cause combined VKD factor deficiency. 2. To determine if turnovers of VKD proteins differ and are impacted by a second site of VKD protein-carboxylase interaction. We will determine whether the VKD proteins factor X and prothrombin are carboxylated with equal efficiencies, and whether having two sites of VKD protein-carboxylase interaction impacts efficiency. 3. To test our hypothesis that post-translational carboxylase modifications are important to VKD protein turnover. Sites of post-translational modification in the carboxylase will be identified and mutated to determine if they impact VKD protein turnover. These studies will make important contributions to understanding carboxylation, which will be significant for developing superior anticoagulants and for producing VKD proteins for therapies in hemophilia and sepsis. Lay abstract. Vitamin K in the diet is used to activate a set of factors critical to blood clotting, and therefore it is important to understand how they become activated. The studies will impact the development of anticoagulants and the production of therapeutic proteins for treating hemophilia and septic shock.